Upload
asher-parker
View
220
Download
3
Tags:
Embed Size (px)
Citation preview
Nano-tech Devices
Functional Surfaces:
chips, membranes, arrays
Signals:
getting information in and out
Actuation:
control of behavior
Hydrophobins
• Proteins excreted by fungi
• Function in growth and
development
• About 100 amino acids
• 8 conserved cysteine
residues
• Self-assemble at
hydrophobic - hydrophilic
interfaces
• Assemblages - very stable
Functional SurfacesSignalsActuation
Lateral force image
•light = high lateral force
Topographic Image
•light = raised surface
- hydrophobin + hydrophobin
Functional SurfacesSignalsActuation
ATR-FTIR
1700 1680 1660 1640 1620 1600
Inte
nsity
Wavenumber (cm-1)
190 200 210 220 230 240 250-2
-1
0
1
2
Elli
ptic
ity x
10-4
(deg
cm2 dm
ol-1
)
Wavelength (nm)
Circular Dichroism
-helix -sheet -turn random coil
Soluble 23 41 16 20
At air-water interface 16 65 9 10
At hydrophobic surface 33 36 17 14
Functional SurfacesSignalsActuation
Molecules exchange between oligomers
0 200 400 600 8001
2
3
4
5
6
7
Flu
ores
cenc
e in
tens
ity (
a.u.
)
Time (min)
TFA/dansyl-SC3+TFA/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3 TFA/dansyl-SC3
Functional SurfacesSignalsActuation
0 200 400 600 8001
2
3
4
5
6
7
Flu
ores
cenc
e in
tens
ity (
a.u.
)
Time (min)
TFA/dansyl-SC3+TFA/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3 TFA/dansyl-SC3
Molecules exchange between oligomers
Functional SurfacesSignalsActuation
SC3 associates in solution and dissociates on a hydrophobic surface
Wavelength (nm) Wavelength (nm)
Flu
ores
cen
ce (
a.u
.)
Flu
ores
cen
ce (
a.u
.)
400 450 500 550 600 6500
1
2
3
4
5
TFA/DX-SC3 TFA/DX-SC3+TFA /DAB-SC3 TFA/DX-SC3+TFA/DAB-SC3+Teflon
400 450 500 550 600 6500
1
2
3
4
5
TFA/dansyl-SC3 TFA/dansyl-SC 3/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3/Teflon
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1
TFA/dansyl-SC3/dabcyl-SC3
TFA/dansyl-SC3/dabcyl-SC3 +Teflon
Rel
ativ
e fl
uor
esce
nce
dabcyl-SC3/(dansyl-SC3+dabcyl-SC3)
Soluble state
-helical state
Teflon
Teflon
Functional SurfacesSignalsActuation
SC3 in -sheet state clusterson a hydrophobic surface
Functional SurfacesSignalsActuation
400 450 500 5500
2
4
6
8
10
12
14
TFA/dansyl-SC3/Teflon TFA/dansyl-SC3/Teflon/65C Add 0.1% Tween80, 15 h
Wavelength (nm)
Flu
ores
cen
ce (
a.u
.)
-helical state
Teflon
-sheet state
Teflon
heating, detergent
600 650
Or low pH
Surface-induced folding of sulfite treated SC3
• Native SC3
• Reduced and reacted form - stable in solution
• Refolding on hydrophobic surface
• Reformation of disulfides by air oxidation
Functional SurfacesSignalsActuation
soluble
-helix form
-sheet form
very fastfastmediumvery slow
Deuterium Exchange Rates vs Structural State
Functional SurfacesSignalsActuation
Engineering Surface Permeability
Goal: control ion permeability through changes in ion selectivity and changes in gating properties
Functional SurfacesSignalsActuation
Pore forming molecules mediating ion fluxes
across (biological) membranes
Ion channels are not mere nano tubes but are characterized by:
- ion selectivity
- gating (opening and closing)
Protein Ion Channels
Functional SurfacesSignalsActuation
In the context of biosensor technology
Ion channels are signal amplifiers:
Channel opening results in a flow of ions as large as 108 per second
Ion channels are signal transducers:
A chemical signal (binding event of the target molecule) can be transduced into an electric current
‘closed’ ‘open’
ligandtarget molecule
Functional SurfacesSignalsActuation
What determines the selectivity of an ion channel?
- size of the permeant ion species
- charge of the permeant ion species
- combination of both
- atomic arrangement in that part of the protein
responsible for the ion selectivity
Ion Channel Selectivity
Functional SurfacesSignalsActuation
Example: L-type Ca2+ channels
Selectivity filter comprises 4 negatively charged glutamates:
-OC-C-CH2-CH2-COO-
I HN I
glutamate (E)
R-COO- -
OOC-R
Ca2+
Ca2+
R-COO-
-OOC-R
EEEE locus
Functional SurfacesSignalsActuation
40 Å+ -
Functional SurfacesSignalsActuation
Switch the essentially non-selective porin (OmpF) into a calcium-selective ion channel by mimicing the dielectric environment found in Ca2+ channels
Goal
Strategy
Use site-directed mutagenesis to put in extra glutamates
and create an EEEE locus in the selectivity filter of OmpF
Site-directed
mutagenesis
R132
R82E42
E132
R42 A82
Wild type EAE mutant
E117 E117
D113D113
Functional SurfacesSignalsActuation
PLANAR LIPID BILAYER SET UPrecordings on a single molecule!
OmpF trimer
ions
Trans Cis
OA
Rf
Phospholipid bilayer
-
+
Vcom
Vout
IfIf
IK
IV-converter
Voltage clamp:- voltage is set- current is measured
Functional SurfacesSignalsActuation
-100 -50 50 100
-150
-50
50
150
ECa
WT
EAE
Current (pA)
Voltage (mV)
Cis Trans
1 M CaCl2 0.1 M CaCl2
Ca2+
Ca2+
IV-PLOT
Cis Trans Cis Trans
IV-plot EAE: current reverses at equilibrium potential of Ca2+ (ECa),
indicating the channel can discriminate between Ca2+ and Cl-
Zero-current potentialor reversal potential = measure of ion selectivity
Functional SurfacesSignalsActuation
PCa/PCl
WT 2.8AAA 25EAE >100
Ca2+ over Cl- selectivity (PCa/PCl)recorded in 1 : 0.1 M CaCl2
SUMMARY OF RESULTS (1)
Conclusions:
- Taking positive charge out of the constriction zone (= -3, see control mutant AAA) enhances the cation over anion permeability.
- Putting in extra negative charge (= -5, see EAE mutant) further increases the cation selectivity.
Functional SurfacesSignalsActuation
PCa/PNa
WT 2.2AAA 3.7EAE 4.2
Ca2+ over Na+ selectivity (PCa/PNa)recorded in 0.1 M NaCl : 0.1 M CaCl2
SUMMARY OF RESULTS (2)
Conclusion:
- Compared to WT, EAE shows just a moderate increase of the Ca2+ over Na+ selectivity.
- To further enhance PCa/PNa may require additional negative charge and/or a change of the ‘dielectric volume’.
Functional SurfacesSignalsActuation
O-1/2
Na+
Ca2+
Ca2+
‘Electric stew’ of Nonner et al.with imposed electroneutrality
- Selectivity filter is a dielectric volume rather than a rigid molecular structure
- ‘Goodness of fit’, selectivity, determined by a proper crowding, it takes twice as much Na+ than Ca2+ to compensate the -4e charge of 8 O’s
‘GOOD’ ‘TOO CROWDED’
Na+
Na+Na+
O-1/2
O-1/2
O-1/2
O-1/2 O-1/2
O-1/2 O-1/2
O-1/2
O-1/2
O-1/2
O-1/2
O-1/2
O-1/2O-1/2
O-1/2
Functional SurfacesSignalsActuation
Dynamic Control of Permeability
Goal: To put permeability under control of external signals – pH– pressure– temperature – redox potential – electric and magnetic fields – ultrasound – light
Functional SurfacesSignalsActuation
pH-Induced Channel Switching
15.6 15.8 16.0 16.2 16.40
1
2
3
4
5
6
Abu
ndan
ce (
a.u)
Molecular Mass (kDa)
MscL.BI
MscL
OH
O
BrN
NH
2-Bromo-3-(5-imidazolyl)propionic acid
Functional SurfacesSignalsActuation
OH
O
BrN
NH
2-Bromo-3-(5-imidazolyl)propionic acid
N N N N
NS
N
O
N
NH2
N NH2
pK a= 5.97pK a= 5.19 pK a= 5.68 pK a= 6.02
pK a= 5.4 pK a= 6.62 pK a= 6.82 pK a= 9.25
pH-Responsive Channel Switching
Functional SurfacesSignalsActuation
(pA
)
0
50
100
pH 7.2
N
(pA)
0
100 pH 5.2
HN
+
pH-sensitive channel openings
Functional SurfacesSignalsActuation
pH-mediated Drug Release from Proteoliposomes
Circulating Liposome Targeted Liposome
Functional SurfacesSignalsActuation
Light-Responsive Channel Proteins
S SHO
O OBr
UV
VisSS
O
HO
O
Br
200 300 400 500 600 7000.0
0.1
0.2
0.3
0.4
0.5
0.6
Abs
orba
nce
Wavelength (nm)
Open Closed
Functional SurfacesSignalsActuation
Acknowledgments
Biomade• M.de Vocht • X. Wang • R. Friesen• H. Meidema • W. Meijberg • A. Sagiroglu
Rush Medical College• B. Eisenberg• J. Tang
U. Of Miami School of Medicine • W. Nonner• D. Gillespie
U. Of Groningen• B. Poolman • B.Feringa • J.van Esch • H. Wosten • J. Wessels • I. Reviakine • W. Bergsma-Schutter• A. Brisson
École Polytechnique Fédérale de Lausanne
• P. Ulrich• H. Vogel